The analysis of plasma/serum samples generated from in vivo studies of therapeutic proteins is of interest in the biopharmaceutical industry. The conventional ELISA approach has been used for over 25 years and has several limitations. The ELISA require high quality custom reagents that can take several months to generate and the assay optimization can take an additional number of months. Thus, ELISA has a long assay development time which is a limitation in both the early discovery stage and the development stage of protein-based drugs (Murray et al (2001) J. Imm. Methods 255:41-56; Kirchner et al (2004) Clin. Pharmacokinetics 43(2):83-95). Suitable ELISA reagents and assay conditions may not be possible in some cases due to the highly custom binding requirements for each protein therapeutic. Another limitation of ELISA is that reagents may bind non-specifically with plasma/serum proteins; matrix interference is a common phenomenon. Protein quantification by mass spectrometry on the other hand is highly specific and therefore matrix interference is rare compared to ELISA. Development of ELISA assays can be labor-intensive and require complex, specific reagents. ELISA is also sensitive to matrix interferences and cross-reactivity of antibodies. ELISA measures analyte concentration indirectly using binding properties. These many variables make ELISA methods of protein quantification challenging to develop and transfer to other laboratories with robust performance. On the basis of these differences, mass spectrometry is an orthogonal method to ELISA. Mass spectrometry methods of protein quantification, LC-MS/MS in particular, do not require custom reagents and generally yields faster assay development. In addition, Mass spectrometry is less subject to matrix interferences and provides generic assay conditions which are highly specific and can be multiplexed and automated. The high specificity of mass spectrometry measures analyte concentration using intrinsic physical chemical properties of the analyte, i.e. mass and fragmentation pattern. The robust format allows ready lab-to-lab transfer, a significant advantage for approved antibody therapies. A general methodology for quantifying proteins by mass spectrometry is trypsin digestion of the intact protein. The resulting peptides are analyzed by mass spectrometry by introducing corresponding stable isotope labeled internal standards at a fixed concentration.
Recent advances in peptide and protein analysis by mass spectrometry (MS) are due to the developments in front-end gas phase ionization and introduction techniques such as electrospray ionization (ESI), and matrix-assisted laser desorption ionization (MALDI, US 2003/0027216), as well as improvements in instrument sensitivity, resolution, mass accuracy, bioinformatics, and software data deconvolution algorithms (“Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications”, Cole, R. B., Ed. (1997) Wiley, New York; “Modern Protein Chemistry: Practical Aspects”, Howard, G. C. and Brown, W. E., Eds. (2002) CRC Press, Boca Raton, Fla., p. 71-102; Martin et al (1997) Cancer Chemother. Pharmacol. 40:189-201; WO 03/046571; WO 03/046572).
Liquid chromatography-tandem mass spectrometry is a powerful tool for protein analysis and quantitation in very complex matrices like plasma/serum samples. Since peptides resulting from the digestion of the protein of interest and other plasma/serum proteins may have the same or similar nominal mass, the second dimension of MS fragmentation often provides a unique fragment of a peptide of interest. The combination of the specific parent peptide and the unique fragment ion is used to selectively monitor for the molecule to be quantified. Such approach is termed “Multiple reaction monitoring” (MRM), also referred to as Selected Reaction Monitoring (SRM), which is a commonly used mode for protein quantitation.
Electrospray ionization (ESI) provides for the atmospheric pressure ionization (API) of a liquid sample. The electrospray process creates highly-charged droplets that, under evaporation, create ions representative of the species contained in the solution. An ion-sampling orifice of a mass spectrometer may be used to sample these gas phase ions for mass analysis. The response for an analyte measured by the mass spectrometer detector is dependent on the concentration of the analyte in the fluid and independent of the fluid flow rate.